Bovine material detection lateral flow ELISA test strip test kit and its application in a kind of food and feedstuffTechnical field
The invention belongs to the PCR nucleic acid amplification of cattle source DNA and lateral flow are exempted from molecular biology and field of immunology, food and feedstuffThe methods for making and using same of epidemic disease colloidal gold strip test kit.
Background technology
For preventing the added beef in animal feed of mad cow disease (bovine spongiform encephalopathy, BSE), Os Bovis seu BubaliPropagate, European Union in 1994 and the U.S. in 1997 formulated at 1994 and 1997 respectively and forbids adding ruminant in the feedstuff of ruminantThe regulation of endogenous binding protein, this ban is expanded to and forbids to being used for the finished mammal of edible detoxification, birds by European Union in 2000With fish protein composition.The Ministry of Agriculture of China promulgated in 2004《Feed products from animal sources safety and sanitation management method》In provide againstUsing feed products from animal sources (except milk and milk productses) in ruminant feed, but artificial incorporation ruminant is (main in animal feedIt is cattle, sheep) phenomenon of meat and bone ware happens occasionally.
In recent years, on domestic and international food products market, the phenomenon of mixing the spurious with the genuine such as false Carnis caprae seu ovis, false beef emerges in an endless stream, and animal derived food doping is adulteratedThe frightened gallbladder of common people's heart is increasingly allowed to quiver etc. taking place frequently of various food safety affairs.At the beginning of 2013, " the Equus caballus (L.) disturbance " in Europe grows in intensity, and makesObtain consumer's " hearing horse complexion changed "." flesh of fish disturbance " American-European afterwards and sounded the alarm bell of food safety again.Food to be ensured and feedstuffSafety, need sensitive, convenient, accurate detection method to provide basis.
In traditional feedstuff, Animal components identification is mainly passed through microscope inspection, the immunological method based on protein detection and is based on nucleic acidAll kinds of PCR method of detection, there have been developed near infrared detection method (NIRS) in recent years.The various animal derived components mixing in feed ingredient are in high temperatureIt is susceptible in process and the course of processing destroy, between the protein sequence of different genera animal, difference is little, it becomes difficult to distinguish, patent of invention" SDS-PAGE method differentiates animal derived composition in meat and meat productss " (publication number：CN 102213720 A) provide a kind of with proteinSDS-PAGE electrophoretic identification pig, cattle, sheep, chicken, the method for the animal proteinum of fish, but the protein electrophoresises of separate sources and processing modeBands of a spectrum are different, bring the obstacle that result differentiates, are more difficult to implement for the animal component in feedstuff.
Because the degeneracy feature of gene coded sequence and the presence of non-coding sequence, nucleic acid becomes the target being more easy to detect difference compared with albumen and dividesSon, particularly mitochondrial DNA, copy number is high, there are interspecies differences, the analysis to its differential fragment is main authentication method, this kind ofMethod is high because of sensitivity, and specificity is good, takes and quickly grows less, main method includes commonly/fluorescence PCR method and gene chip skillArt.When carrying out the species discrimination of species with mtdna sequence, conventional region includes the Cytochrome b of Codocyte pigment BGene (cytB), small subunit ribosome 12S RNA coded sequence (12S ribosomal RNA), cytochrome C subunit I (cytochromeC oxidase subunit I) encoding gene coxI, the coding atp8 and atp6 gene of ATP8 and ATP6 and control zone D-loop pieceSection.The sequence variations in these regions are moderate, have certain conservative in planting, embody certain intermediate diversity again.
Fluorescent quantitative PCR technique is a kind of highly sensitive nucleic acid detection and quantitative approach, and ultimate principle is anti-in real-time fluorescence quantitative PCRYing Zhong, introduces a kind of fluorescent chemical, and with the carrying out of PCR reaction, PCR product constantly accumulates, and fluorescence signal intensity is also etc.Ratio increases.Fluorescent quantitative PCR technique makes qualitative detection step the step that can quantify, and has sensitivity height, specificity and reliabilityProperty higher, enable multiple reaction, high degree of automation, nonstaining property, there is the features such as real-time and accuracy, particularly rely on probeIn conjunction with TaqMan method, be widely used to the fields such as molecular biology research and medical research at present.In species identification application aspect,Domestic issued national standard or industry standard cover the mammals such as cattle, sheep, deer, camel, horse, donkey, pig, rabbit, Canis familiaris L. and fishOr aquatile.In view of the importance of ruminant, patent of invention " fluorescence PCR detection reagent of discriminating ruminant derived component and preparationMethods and applications " (publication number：CN 101659996A) describe a kind of fluorescence PCR method that can simultaneously differentiate cattle, sheep and goat, with threeBar probe carries out animal origin examination, patent of invention " donkey or the inspection of donkey derived component real-time fluorescence PCR of Publication No. CN 102311999 ASurvey method and detection primer and probe " provides the fluorescence PCR method differentiating donkey origin components with particular probe, and Publication No. CNThe patent of invention " horse or horse derived component real-time fluorescence PCR detection method and detection primer and probe " of 102311998 A then provides onePlant the fluorescence PCR method of identification Malaysia derived components.A kind of patent of invention " discriminating side of animal derived materials of Publication No. CN101196463Method " provides one group and can differentiate the vertebrate primer sequence such as cattle, sheep, pig, horse, chicken and Standard PCR-gel electrophoresis method, is similar to, in terms of the identification of poultry source, patent of invention " a pair of goose derived component PCR detection primer " (publication number：CN 102337337A a kind of method identified with Standard PCR, Publication No. CN 101270392A patent of invention) are described with reference to agarose gel electrophoresiies" amplimer of the PCR-mtDNA total avian composition of detection and its detection kit and using method " is then further provided and is drawn with a pairThe method of the multiple poultry derived mitochondrial DNA of analyte detection, in said method, real-time quantitative PCR detection strong to device dependence it is difficult to realExisting Site Detection, conventional PCR augmentation detection needs to carrying out image acquisition and analysis after product gel electrophoresis, and the time of expending is longer, and difficultTo ensure the sensitivity of detection.
By the method for immunology detection being transplanted the detection efficiency that can effectively improve nucleic acid product in nucleic acid product detection.By by PCRThe specific protein of primer mark that amplification uses or compound can introduce in its amplified production and include two kinds of compound/protein labelings simultaneouslyNucleic acid complexes, the product of this labelling can be detected by way of antibody or part are with similar double-antibody sandwich, this processSimilar conventional immuno-gold labeling detection technique, this method makes in the method for single nucleotide polymorphism and constant-temperature amplification target geneWith, the particularly identification to pathogenic microorganism or disease association gene, Publication No. CN 102134596 A patent of invention-" one kind is based onThe method that nucleic acid laterally flows ELISA test strip single nucleotide polymorphism " carries out the detection of single nucleotide polymorphism in this approach.And Publication No.The patent of invention "-mono- increasing listeria spp nucleic acid chromatography detection kit and its detection method and application " of CN 102520172 A describes one kindIt is respectively adopted the cow genome group detection method of biotin and digoxigenin labeled primer.Therefore class method only judges to the presence or absence of amplified production,, not being confirmed, the interference factor therefore in amplified reaction is some more, and the common false-positive reason that causes includes to the molecular weight of product：Non-Abnormal renaturation between specific amplification, primer dimer, amplified production, solves primer dimer, the interference causing of extremely annealing can be fromThe selection of PCR polymerase, primer sequence optimization, dNTP substrate, the several aspect of control of crossover process are carried out.Selection has thermal starting(Hot-start) archaeal dna polymerase of function can substantially reduce the formation of primer dimer and non-specific amplification product.In primer optimization sideFace, patent of invention-" partly the reducing its dimeric method with sequence primer a pair " of Publication No. CN 102146432 A describes one kind and existsThe primer design method with short palindrome for the primer 5 ' end, recirculation under this primer room temperature, it is to avoid form heterodimer, Publication No.The patent of invention of CN 102719547 A-" the real-time fluorescence quantitative PCR reagent of detection HER2 gene expression dose " also uses similarMethod is used for the amplification of real-time quantitative PCR；The patent of invention of Publication No. CN 101842494 A-" reduce heterodimeric using chimeric primersBody is formed " describe a kind of method that use chimeric primers are expanded；In the optimization to reaction substrate, publication number CN 101171343APatent of invention " 3 ' modified oligonucleotides containing false iso-cytosine nucleobase derivative and its application as primer or probe " provideA kind of nucleotide that special modification be used method to reduce primer dimer formation as substrate, using probe or introduce internal control probe and also may be usedTo reduce the interference of non-specific amplification, the patent of invention of Publication No. CN 101957373 A-" a kind of add internal control nucleic acid to pathogen nucleic acidThe method carrying out half-quantitative detection " to reduce interference using internal control probe, in said method, the use of thermal starting technology and cyclisation primer is logicalMethod, remaining method or the substrate and the substrate that employ special synthesis of row, or need to introduce second crossover process by probe, allThe complexity of detection process, particularly probing procedure can be increased, lose the convenience of test strips method because needing insulating process.
In design of primers of the present invention, on keeping Specific basal, the free energy of 5 ' and 3 ' end annealing in primer pair and primer is carried outSuitable region is selected to avoid dimeric formation after assessment, long with suitable amplicon for the dimeric disturbed condition of ELISA test strip methodDegree, cooperation launches the concentration optimization of detergent and denaturant in buffer, it is possible to achieve primer dimer and non-specific amplification product effectively clearRemove.
Content of the invention
The nucleic acid molecules of biological micromolecule or compound label can be identified by the antibody specificity of this small molecule or compound, thus passing throughImmunological method detects.The common molecule that can be used for labeled nucleic acid molecule includes hapten molecule biotin (Biotin), digoxin(Digoxin), luminescent dye molecule rhodamine (RBITC), Fluorescein isothiocyanate (fluorescein isothiocyanate, FITC), Cy3,Cy5 etc..In above-mentioned labelling molecule, digoxin, Fluorescein isothiocyanate are all easily obtained special antibody, and biotin molecule and its part-Streptavidin has the binding characteristic of high special, and therefore this several molecule may be selected for labelling and the immunology detection of nucleic acid molecules.It is contemplated that using antigen or the single-stranded oligonucleotide of hapten small molecule tags as primer, to target nucleic acid specific amplification to be checked, the answering of formationAdduct molecule is provided simultaneously with two kinds of antigens or hapten-marked, immunogenic, and this complex can use and specific antibody or sepcific ligands knotClose, realize the immune colloid gold detection of nucleic acid amplification product.
Therefore, on the one hand, the present invention provides a kind of test strips method to detect the detection technique of bovine material in food and feedstuff, main inclusion：
1) two unique amplimers are designed, wherein forward primer Bos-ATP-F has following sequence：5′-GCCATATACTCTCCTTGGTGAC-3 ', with antigen or hapten molecule biotin (Biotin), Fluorescein isothiocyanateOr one of digoxin (Digoxin) labelling (FITC)；The sequence of downstream primer Cat-T-ATP-R be 5 '-TAGTAGGCTTGGGAATAGTACG-3 ', with a kind of labelling different from forward primer,；Under suitable amplification condition, can expandIncrease the DNA fragmentation that bovine mitochondrial gene group atp8-atp6 region one segment length is 270bps；When no tested nucleic acid-templated in the presence of, no specialProperty nucleic acid fragment amplified production；
2) by a kind of universal antibody of standard, such as anti-FITC antibody is crosslinked with colloid gold particle, forms coated antibody in particle surface；
3) by the antibody of another kind of labelling molecule or part, the part-streptavidin molecule of such as anti digoxin antibody or biotin molecule is fixed with linearDetection line is formed on film；
4) when there are specific amplification products to be checked, because of the effect of upstream and downstream primer, simultaneous with above-mentioned three kinds of labels in amplified productionTwo kinds, form the complex of labelling molecule 1- amplified production-labelling molecule 2；
5) by step 4) mark 1- amplified production-mark of being formed 2 and the antibodies of the mark 1 of colloid gold particle pan coating, formed anti-Mark 1 antibody-mark 1- amplified production-mark 2 coloured particle complex；
6) by step 5) the coloured particle complex that obtains travels up to, by capillarity, the antibody being coated with mark 2 in the solution along fibrous membraneOr on the lines of part, complex deposits because the antibody (part) with mark 2 combines, and is trapped in detection line, forms naked eyes visibleColored line, be judged to the positive；7) when specific amplification products do not exist, there are not above-mentioned steps 4) -6) it is impossible to form mark 1-Amplified production-mark 2 complex, also shape cannot be deposited on the antibody (part) of mark 2 in detection line, is not formed visibleBand, is judged to feminine gender.
On the other hand, present invention also offers being used for the developping solution of nucleotide sequence detection, this developping solution can reduce primer dimer to experimentThe interference of result；
On the other hand, the invention provides being used for the test strips of the quick detection of food borne pathogenic microorganism, it includes in the liner with adhesive sticker,Have in order：1：Absorbent filter pad；2：Granule (the gold colloidal that adds lustre to of conjugate pad, the antibody with the first nucleic acid markers or partOr latex)；3：Lateral chromatography substrate (nitrocellulose filter or nylon membrane)；4：Detection band, with the antibody of second nucleic acid markers；5：Quality control band, with can be in conjunction with the antibody of specific Species origin antibody；6：Substrate；7：Adsorptive pads.
On the other hand, present invention also offers being used for detecting the universal standard test strips of nucleic acid amplification product.
Finally, present invention also offers above-mentioned detection method and test strips in food and feedstuff calf-derived Cyclospora detection in purposes.
Explanation of technical terms used in description of the invention：
Primer：The starting material of DNA synthesis.Generally a pair of single stranded oligonucleotide, after hybridizing with template, DNA synthesis is opened from its 3 ' endBegin.
Labelling：The method that detectable signaling molecule (such as hapten, fluorescence, radioactivity etc.) is coupled with single stranded oligonucleotide phase.
Hybridization：The DNA refering in particular to complementation single-stranded forms duplex structure by base pairing.
Launch：PCR primer through amplified reaction launch buffer chromatography effect under from test strips adsorptive pads bottom start to detection line,The process of nature controlling line direction movement.
Nucleic acid：DNA (deoxyribonucleic acid) (DNA) and the common name of ribonucleic acid (RNA).
Antigen and hapten：Possesses immunogenic material.It is usually macro-molecular protein or cellular component.But some small molecules also possessImmunogenicity, is referred to as hapten ((Hapten).Hapten is commonly used for label probe.
Antibody：The protein molecule being combined with antigen or hapten specificity.
Complex：The conjugate formed by two or more molecular specificity.
Primer dimer：During polymerase chain reaction (PCR), because of primer pair between or wall scroll primer mutually anneal the two of formationPolymer molecular, the dimer being made up of two different primers is heterodimer, because causing with two kinds of labellings and plant antibody (part)Combination and cause false positive, the dimer being formed by single primer annealing is homodimer, only carries a kind of labelling without causing false sunProperty, but excessive dimer formation can reduce amplification efficiency.
Immunity test strip：Medical tool for quick detection.It is also called colour generation membrane chromatographic.
Nucleic acid polymerase：The enzyme of nucleic acid long-chain.It is divided into archaeal dna polymerase and RNA polymerase two big class.
Beneficial effect：
Because the present invention is with the genetic fragment of the primer amplified suitable length of labelling, ensure that the high degree of specificity of detection, thus ensureingThe accuracy of testing result.Avoid the flow process such as amplified production dilution and probe hybridization in place of the novelty of the present invention, maintain immune colloid goldThe advantages of (test strips) technology is directly perceived, quick, convenient, ripe, cheap, but also with PCR amplification method height sensitivity, high degree of specificityFeature, therefore, the method for the present invention is a kind of effective cattle source animal component detection method.
As the current techique platform of nucleic acid amplification product detection, the method for the present invention and its test strips can be widely applied to food andThe detection of the identification of animal derived components and food borne pathogenic microorganism in feedstuff, before farming and animal husbandry, customs inspection quarantine are all widely usedScape.Though having the animal sources Components identification method of multiple PCR-baseds, its design of primers and product fragment to be suitable for sonde method or gel electricity at presentSwimming, and the interference reducing primer dimer and non-specific renaturation in colloidal gold strip method is removed and lacks necessary consideration, solutionAnd it is inapplicable.Present invention aim at providing a kind of PCR primer, amplification and detection detecting cattle source animal component based on immune colloidal gold methodMethod, and the application of the method.Other common species, such as cattle, sheep, pig, donkey, rabbit, camel, duck, goose, chicken, fox, ermine,The identification of deer etc. can be completed using similar method.
The invention of nucleic acid test strip detection method will greatly simplify the detection program after nucleic acid amplification.The simple and clear, directly perceived (inspection of result interpretationMeasure meaning result such as accompanying drawing 2).
In the apllied food of this patent and feedstuff, calf-derived Cyclospora Rapid detection test strip technology detects as after a kind of new nucleic acid amplificationTechnology, has advantages below：
Simple to operate：Sample after only needing to nucleic acid amplification directly drips to can be easy to it is not necessary to professional's operation in nucleic acid reagent detection versionPromote；
Quickly：Sentence read result after detecting 5 minutes；
Sensitive：Compared with agarose gel electrophoresiies, detection sensitivity improves nearly 100～200 times；
Specificity is high：Due in detection process plus use for specificity labeled primers, make result more accurate；
Low price：Expense is significantly less than traditional gel electrophoresiss and ELISA detection.
Brief description
Fig. 1 shows the basic structure of the test strips of amplification of nucleic acid sequences product detection of the present invention；
1：Absorbent filter pad；2：The granule that adds lustre to (gold colloidal or latex) of conjugate pad, the antibody with the first nucleic acid markers or part；3：Lateral chromatography substrate (nitrocellulose filter or nylon membrane)；4：Detection band, with the antibody of second nucleic acid markers；5：Quality ControlBand, with can be in conjunction with the antibody of specific Species origin antibody；6：Substrate；7：Adsorptive pads.
Fig. 2 is the testing result schematic diagram of nucleic acid detection test strip；
Fig. 3 is the method showing with embodiment 1, detects the testing result of the PCR- test strips of cattle using specificity labeled primers；
Fig. 4 is the method showing with embodiment 2, using the testing result of the specific PCR-test strips of detection bovine labeled primer；
Fig. 5 is the method with embodiment 2, the sensitivity comparative result of test strips and gel electrophoresis assays after show nucleic acid amplification.
Specific embodiment
Following examples illustrate detection method and its Detection results of the present invention.Embodiment is only used for illustrating, and does not constitute to the present inventionAny restriction of protection domain, protection scope of the present invention illustrates in appending claims.
Embodiment 1
1 materials and methods
Cow genome group DNA
1.2 design of primers
1.3PCR amplification system：
1.3PCR amplification system：
| Template DNA：Cow genome group DNA | 1.0μl |
| PrimerF/R | 0.8μl |
| dNTP | 2.0μl |
| 10X PCRbuffer | 2.0μl |
| HS Taq DNA polymerase | 0.2μl |
| ddH2O | 13.2μl |
| Cumulative volume | 20μl |
Reaction condition：
| 95℃ | 5min |
| 94℃ | 30sec |
| 55℃ | 30sec |
| 72℃ | 30sec |
| 72℃ | 5min |
| Totally 30 circulation | |
Take 3 μ l to take 3 μ L amplified productions for nucleic acid test strip detection simultaneously respectively, be added in 97 μ L developping solutions and carry out test point in samplePad, observed result after 5 minutes.
1.4PCR specificity experiments
Using the PCR reaction system set up to 1：Mus；2：Cattle；3：Rabbit；4：Horse；5：Sheep；6：Canis familiaris L.；7：Pig；8：Cat；9：Chicken；10：Duck；11：Goose；12：NTC (blank：Water) verify its specificity.
2 results
2.1PCR reaction system and condition
Using the HS Taq DNA polymerase of TAKARA company, overall reaction system is 20 μ L.Detected with Bio-Rad PCR instrument,Response parameter is：94 DEG C of 5min, 94 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s are expanded, and proceed to 72 DEG C 5 after 30 circulationsMin, 4 DEG C of preservations.Take 5 μ L products to carry out electrophoresis in 2% agarose gel electrophoresiies containing ethidium bromide, after electrophoresis, determine whether specificityBand.After nucleic acid amplification, test strips and the testing result of gel electrophoresiss are shown in accompanying drawing 4.
2.2 specific test
The PCR method that the present invention sets up has preferable specificity to cow genome group DNA, and other mammals and poultry species etc. are no intersectedReaction.
Embodiment 2
The susceptiveness of nucleic acid test strip detection method, by pcr template with 1/10,1/102, 1/103, 1/104, 1/105, 1/106, 1/107,1/108, 1/109, 1/1010Dilution proportion after, expanded by the PCR system of foundation.
1 materials and methods
Cattle mitochondrial DNA
1.2 design of primers
1.3PCR amplification system：
| Template DNA：Plasmid DNA | 10μl |
| Forward primer | 0.8μl |
| Downstream primer | 0.8μl |
| dNTP | 2.0μl |
| 10X PCR buffer | 2.0μl |
| HS Taq DNA polymerase | 0.2μl |
| ddH2O | 4.2μl |
| Cumulative volume | 20μl |
Reaction condition：
Take 5 μ l for agarose gel electrophoresiies.Gel electrophoresis conditions are：1X tbe buffer liquid, voltage 100V, 30 points of electrophoresis time.SimultaneouslySeparately take 5 μ l to detect for nucleic acid test strip, that is, take 5 μ l sample spot in sample pad, be added in 95 μ L developping solutions and detected, 5 pointsObserved result after clock.
2 results
2.1PCR reaction system and condition
Using the HS Taq DNA polymerase of TaKaRa company, overall reaction system is 20 μ l.Expanded with Bio-Rad T-100PCR instrumentIncrease, response parameter is：94 DEG C of 5min, 94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 30s are expanded, and proceed to 72 DEG C 5 after 30 circulationsMin, 4 DEG C of preservations.Take 5 μ l products to carry out electrophoresis in 2% agarose gel electrophoresiies containing ethidium bromide, after electrophoresis, determine whether specificityBand.After nucleic acid amplification, test strips and the sensitivity comparative result of gel electrophoresiss are shown in accompanying drawing 5, be can be seen that by the result of accompanying drawing 5Compared with traditional gel electrophoresiss, its sensitivity increases by 100～200 times.
GCCATATACTCTCCTTGGTGACATGCCGCAACTAGACACGTCAACATGACTGACAATGATCTTATCAATATTCTTGACCCTTTTTATCATCTTTCAACTAAAAGTTTCAAAACACAACTTTTATCACAATCCAGAACTGACACCAACAAAAATATTAAAACAAAACACCCCTTGAGAAACAAAATGAACGAAAATTTATTTACCTCTTTTATTACCCCTGTAATTTTAGGTCTCCCTCTCGTAACCCTTATCGTACTATTCCCAAGCCTACTA